U.S. patent application number 14/270283 was filed with the patent office on 2014-11-06 for target detection with nanopore.
The applicant listed for this patent is Two Pore Guys, Inc.. Invention is credited to Trevor J. Morin.
Application Number | 20140329225 14/270283 |
Document ID | / |
Family ID | 51023027 |
Filed Date | 2014-11-06 |
United States Patent
Application |
20140329225 |
Kind Code |
A1 |
Morin; Trevor J. |
November 6, 2014 |
TARGET DETECTION WITH NANOPORE
Abstract
Provided are methods for detecting a target molecule or particle
suspected to be present in a sample, comprising (a) contacting the
sample with (i) a fusion molecule comprising a ligand capable of
binding to the target molecule or particle and a binding domain,
and (ii) a polymer scaffold comprising at least one binding motif
to which the binding domain is capable of binding, under conditions
that allow the target molecule or particle to bind to the ligand
and the binding domain to bind to the binding motif; (b) loading
the polymer into a device comprising a pore that separates an
interior space of the device into two volumes, and configuring the
device to pass the polymer through the pore from one volume to the
other volume, wherein the device further comprises a sensor
adjacent to the pore configured to identify objects passing through
the pore; and (c) determining, with the sensor, whether the fusion
molecule or particle bound to the binding motif is bound to the
target molecule or particle, thereby detecting the presence of the
target molecule or particle in the sample.
Inventors: |
Morin; Trevor J.; (Santa
Cruz, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Two Pore Guys, Inc. |
Santa Cruz |
CA |
US |
|
|
Family ID: |
51023027 |
Appl. No.: |
14/270283 |
Filed: |
May 5, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61820083 |
May 6, 2013 |
|
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Current U.S.
Class: |
435/5 ; 422/69;
435/287.1; 435/287.2; 435/7.23; 435/7.32; 436/501 |
Current CPC
Class: |
G01N 33/54366 20130101;
G01N 33/574 20130101; C12Q 1/6869 20130101; G01N 33/5302 20130101;
G01N 27/44791 20130101; G01N 27/4473 20130101; C12Q 1/6869
20130101; G01N 33/536 20130101; G01N 33/54306 20130101; G01N
33/56911 20130101; C12Q 2563/116 20130101; C12Q 2563/131 20130101;
G01N 33/48721 20130101; C12Q 2565/531 20130101; B81C 1/00158
20130101; C12Q 2565/631 20130101; G01N 33/56983 20130101 |
Class at
Publication: |
435/5 ; 436/501;
435/7.32; 435/7.23; 435/287.2; 435/287.1; 422/69 |
International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 33/574 20060101 G01N033/574; G01N 33/569 20060101
G01N033/569 |
Claims
1. A method for detecting a target molecule or particle suspected
to be present in a sample, comprising: (a) contacting the sample
with (i) a fusion molecule comprising a ligand capable of binding
to the target molecule or particle and a binding domain, and (ii) a
polymer scaffold comprising at least one binding motif to which the
binding domain is capable of binding, under conditions that allow
the target molecule or particle to bind to the ligand and the
binding domain to bind to the binding motif; (b) loading the
polymer into a device comprising a pore that separates an interior
space of the device into two volumes, and configuring the device to
pass the polymer through the pore from one volume to the other
volume, wherein the device further comprises a sensor adjacent to
the pore configured to identify objects passing through the pore;
and (c) determining, with the sensor, whether the fusion molecule
bound to the binding motif is bound to the target molecule or
particle, thereby detecting the presence of the target molecule or
particle in the sample.
2. The method of claim 1, wherein the target molecule is selected
from the group consisting of a protein, a peptide, a nucleic acid,
a chemical compound, an ion, and an element.
3. The method of claim 1, wherein the target particle is selected
from the group consisting of a protein complex or aggregate, a
protein/nucleic acid complex, a fragmented or fully assembled
virus, a bacterium, a cell, and a cellular aggregate.
4. The method of claim 1, wherein step (a) is performed prior to
step (b).
5. The method of claim 1, wherein step (b) is performed prior to
step (a).
6. The method of claim 1, further comprising applying a condition
suspected to alter the binding between the target molecule or
particle and the ligand, and carrying out the determination
again.
7. The method of claim 6, wherein the condition is selected from
the group consisting of removing the target molecule or particle
from the sample, adding an agent that competes with the target
molecule or particle, or the ligand for binding, and changing the
pH, salt, or temperature.
8. The method of claim 1, wherein the binding motif comprises a
chemical modification for binding to the binding domain.
9. The method of claim 8, wherein the chemical modification is
selected from the group consisting of acetylation, methylation,
summolation, glycosylation, phosphorylation, and oxidation.
10. The method of claim 1, wherein the polymer is at least one of a
deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a peptide
nucleic acid (PNA), a DNA/RNA hybrid, and a polypeptide.
11. The method of claim 1, wherein the binding domain is selected
from the group consisting of a helix-turn-helix, a zinc finger, a
leucine zipper, a winged helix, a winged helix turn helix, a
helix-loop-helix, and a high mobility group box (HMG-box).
12. The method of claim 1, wherein the binding domain is selected
from the group consisting of locked nucleic acids (LNAs), peptide
nucleic acids (PNAs), transcription activator-like effector
nucleases (TALENs), clustered regularly interspaced short
palindromic repeats (CRISPRs), and aptamers.
13. The method of claim 1, wherein the ligand is selected from the
group consisting of an antibody, an epitope, a hormone, a
neurotransmitter, a cytokine, a growth factor, a cell recognition
molecule, and a receptor.
14. The method of claim 1, wherein the binding domain and the
ligand are linked via an interaction selected from the group
consisting of a covalent bond, a hydrogen bond, an ionic bond, a
metallic bond, a van der Walls force, a hydrophobic interaction,
and a planar stacking interaction, or are translated as a
continuous polypeptide, to form the fusion molecule.
15. The method of claim 1, further comprising contacting the sample
with a detectable label capable of binding to the target molecule
or particle, or the target molecule or particle/ligand complex.
16. The method of claim 1, wherein the polymer comprises at least
two units of the binding motif.
17. The method of claim 1, wherein the polymer comprises at least
two different binding motifs; the sample is in contact with at
least two fusion molecules each comprising a different binding
domain capable of binding to each binding motif and a different
ligand capable of binding to a different target molecule or
particle; and the sensor is configured to identify whether the
fusion molecule bound to each binding motif is bound to a target
molecule or particle.
18. The method of claim 1, wherein the device comprises electrodes
to apply a voltage differential between the two volumes.
19. The method of claim 1, wherein the device comprises an upper
chamber, a middle chamber and a lower chamber, wherein the upper
chamber is in communication with the middle chamber through a first
pore, and the middle chamber is in communication with the lower
chamber through a second pore; wherein the first pore and second
pore are about 1 nm to about 100 nm in diameter, and are about 10
nm to about 1000 nm apart from each other; and wherein each of the
chambers comprises an electrode for connecting to a power
supply.
20. The method of claim 1, wherein the device comprises an upper
chamber, a middle chamber and a lower chamber, wherein the upper
chamber is in communication with the middle chamber through a first
pore, and the middle chamber is in communication with the lower
chamber through a second pore; wherein the first pore and second
pore are about 100 nm to about 10000 nm in diameter, and are about
10 nm to about 1000 nm apart from each other; and wherein each of
the chambers comprises an electrode for connecting to a power
supply.
21. The method of claim 1, further comprising moving the polymer in
a reversed direction after the binding motif passes through the
pore, such as to identify, again, whether the fusion molecule bound
to each binding motif is bound to a target molecule or
particle.
22. A kit, package or mixture for detecting the presence of a
target molecule or particle, comprising: (a) a fusion molecule
comprising a ligand capable of binding to the target molecule or
particle and a binding domain; (b) a polymer scaffold comprising at
least one binding motif to which the binding domain is capable of
binding; and (c) a device comprising a pore that separates an
interior space of the device into two volumes, wherein the device
is configured to allow the polymer to pass through the pore from
one volume to the other volume, and wherein the device further
comprises a sensor adjacent to the pore configured to identify
whether the binding motif is (i) bound to the fusion molecule while
the ligand is bound to the target molecule or particle, (ii) bound
to the fusion molecule while the ligand is not bound to the target
molecule or particle, or (iii) not bound to the fusion
molecule.
23. The kit, package or mixture of claim 22, further comprising a
sample suspected of containing the target molecule or particle.
24. The kit, package or mixture of claim 23, wherein the sample
further comprises a detectable label capable of binding to the
target molecule or particle, or the target molecule or
particle/ligand complex.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) to U.S. provisional application Ser. No. 61/820,083,
filed May 6, 2013, the contents of which are incorporated here by
reference in their entirety.
BACKGROUND
[0002] Detection of nano-scale particles, such as circulating tumor
cells, bacteria and viruses, has immersive clinical utility.
Currently available methods include immunohistochemistry and
nucleic acid-based detection, and cell proliferation is typically
required before a sensitive detection can be carried out.
[0003] Molecular detection and quantitation are also important, and
can be carried out with various methods depending on the type of
the molecule. For instance, nucleotide sequences can be detected by
virtue of their sequence complementarity to a probe or primer,
through hybridization and/or amplification, or in fewer occasions
with a protein that recognize the sequence. A protein, on the other
hand, is commonly detected with an antibody that specifically
recognizes and binds the protein. An enzyme-linked immuno sorbent
assay (ELISA), in this respect, is highly commercialized and
commonly used.
[0004] Methods also exist for detecting or quantitating various
other large or small molecules, such as carbohydrates, chemical
compounds, ions, and elements.
[0005] Methods and systems for highly sensitive detection of
molecules as well as particles, such as tumor cells and pathogenic
organisms, have broad applications, in particular clinically, for
pathogen detection and disease diagnosis, for instance.
SUMMARY
[0006] In one embodiment, the present disclosure provides a method
for assaying whether a target molecule or particle is present in a
sample, comprising: (a) contacting the sample with (i) a fusion
molecule comprising a ligand capable of binding to the target
molecule or particle and a binding domain, and (ii) a polymer
scaffold comprising at least one binding motif to which the binding
domain of the fusion molecule is capable of binding, under
conditions that allow the target molecule or particle to bind to
the ligand and the binding domain to bind to the binding motif; (b)
loading the polymer into a device comprising a pore that separates
an interior space of the device into two volumes, and configuring
the device to pass the polymer through the pore from one volume to
the other volume, wherein the device further comprises a sensor
adjacent to the pore configured to identify objects passing through
the pore; and (c) determining, with the sensor, whether the fusion
molecule bound to the binding motif is bound to the target molecule
or particle, thereby detecting the presence of the target molecule
or particle in the sample.
[0007] In some aspects, the target molecule is selected from the
group consisting of a protein, a peptide, a nucleic acid, a
chemical compound, an ion, and an element.
[0008] In some aspects, the target particle is selected from the
group consisting of protein complexes and protein aggregates,
protein/nucleic acid complexes, fragmented or fully assembled
viruses, bacteria, cells, and cellular aggregates.
[0009] In some aspects, step (a) of the method for assaying whether
a target molecule or particle is present in a sample is performed
prior to step (b). In some aspects, step (b) is performed prior to
step (a).
[0010] In some aspects, the method further comprises applying a
condition suspected to alter the binding between the target
molecule or particle and the ligand and carrying out the
determination again. In some aspects, the condition is selected
from the group consisting of removing the target molecule or
particle from the sample, adding an agent that competes with the
target molecule or particle or the ligand for binding, and changing
the pH, salt, or temperature.
[0011] In some aspects, the binding motif comprises a chemical
modification for binding to the binding domain. In some aspects,
the chemical modification is selected from the group consisting of
acetylation, methylation, summolation, glycosylation,
phosphorylation, and oxidation.
[0012] In some aspects, the polymer comprises a deoxyribonucleic
acid (DNA), a ribonucleic acid (RNA), a peptide nucleic acid (PNA),
a DNA/RNA hybrid, or a polypeptide.
[0013] In some aspects, the binding domain is selected from the
group consisting of a helix-turn-helix, a zinc finger, a leucine
zipper, a winged helix, a winged helix turn helix, a
helix-loop-helix and an HMG-box.
[0014] In some aspects, the binding domain is selected from the
group consisting of locked nucleic acids (LNAs), PNAs,
transcription activator-like effector nucleases (TALENs), clustered
regularly interspaced short palindromic repeats (CRISPRs), and
aptamers.
[0015] In some aspects, the ligand is a protein. In some aspects,
the ligand is selected from the group consisting of an antibody, an
epitope, a hormone, a neurotransmitter, a cytokine, a growth
factor, a cell recognition molecule, and a receptor.
[0016] In some aspects, the binding domain and the ligand are
linked, via a covalent bond, a hydrogen bond, an ionic bond, a
metallic bond, van der Walls force, hydrophobic interaction, or
planar stacking interaction, or are translated as a continuous
polypeptide, to form the fusion molecule.
[0017] In some aspects, the method further comprises contacting the
sample with a detectable label capable of binding to the target
molecule or particle or target/ligand complex.
[0018] In some aspects, the polymer comprises at least two units of
the binding motif.
[0019] In some aspects, the polymer comprises at least two
different binding motifs: the sample is in contact with at least
two fusion molecules each comprising a different binding domain
capable of binding to each binding motif and a different ligand
capable of binding to a different target molecule or particle; and
the sensor is configured to identify whether the fusion molecule
bound to each binding motif is bound to a target molecule or
particle.
[0020] In some aspects, the device comprises electrodes to apply a
voltage differential between the two volumes.
[0021] In some aspects, the device comprises an upper chamber, a
middle chamber and a lower chamber, wherein the upper chamber is in
communication with the middle chamber through a first pore, and the
middle chamber is in communication with the lower chamber through a
second pore.
[0022] In one aspect, the first pore and second pore are about 1 nm
to about 100 nm in diameter. Such pores can be suitable for
detecting molecules such as proteins and nucleic acids. In one
aspect, the first pore and second pore are as large as about 50,000
nm in diameter, which can be suitable for detecting larger
particles such as tumor and bacterial cells.
[0023] In some aspects, the pores are about 10 nm to about 1000 nm
apart from each other.
[0024] In some aspects, each of the chambers comprises an electrode
for connecting to a power supply.
[0025] In some aspects, the method further comprises moving the
polymer at a reversed direction after the binding motif passes
through the pore, such as to identify, again, whether the fusion
molecule bound to each binding motif is bound to a target molecule
or particle.
[0026] Also provided are kits, packages or mixtures for detecting
the presence of a target molecule or particle. In some aspects, the
kit, package or mixture comprises (a) a fusion molecule comprising
a ligand capable of binding to the target molecule or particle and
a binding domain, (b) a polymer scaffold comprising at least one
binding motif to which the binding domain is capable of binding,
(c) a device comprising a pore that separates an interior space of
the device into two volumes, wherein the device is configured to
allow the polymer to pass through the pore from one volume to the
other volume, and wherein the device further comprises a sensor
adjacent to the pore configured to identify whether the binding
motif is (i) bound to the fusion molecule while the ligand is bound
to the target molecule or particle, (ii) bound to the fusion
molecule while the ligand is not bound to the target molecule or
particle, or (iii) not bound to the fusion molecule.
[0027] In some aspects, the kit, package or mixture further
comprises a sample suspected of containing the target molecule or
particle. In some aspects, the sample further comprises a
detectable label capable of binding to the target molecule or
particle or the ligand/target molecule or particle complex.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Provided as embodiments of this disclosure are drawings
which illustrate by exemplification only, and not limitation,
wherein:
[0029] FIG. 1 illustrates the detection of a target molecule or
particle with one embodiment of the presently disclosed method;
[0030] FIG. 2 provides the illustration of a more specific example
in which a double-stranded DNA is used as the polymer scaffold and
a human immunodeficiency virus (HIV) envelop protein as the ligand,
for the detection of an anti-HIV antibody;
[0031] FIG. 3 shows that a binding between a target molecule or
particle and a fusion molecule can be detected since it has a
different current profile compared to the fusion molecule alone or
the DNA alone, when passing through a nanopore;
[0032] FIG. 4 illustrates the multiplexing capability of the
present technology by including different binding motifs in the
polymer scaffold; and
[0033] FIGS. 5(I)-(III) illustrate a nanopore device with at least
two pores separating multiple chambers.
[0034] Specifically, FIG. 5(I) is a schematic of a dual-pore chip
and a dual-amplifier electronics configuration for independent
voltage control (V.sub.1 or V.sub.2) and current measurement
(I.sub.1 or I.sub.2) of each pore. Three chambers, A-C, are shown
and are volumetrically separated except by common pores.
[0035] FIG. 5(II) is a schematic where electrically, V.sub.1 and
V.sub.2 are principally applied across the resistance of each
nanopore, by constructing a device that minimizes all access
resistances to effectively decouple I.sub.1 and I.sub.2.
[0036] In FIG. 5(III), competing voltages are used for control,
with arrows showing the direction of each voltage force.
[0037] Some or all of the figures are schematic representations for
exemplification; hence, they do not necessarily depict the actual
relative sizes or locations of the elements shown. The figures are
presented for the purpose of illustrating one or more embodiments
with the explicit understanding that they will not be used to limit
the scope or the meaning of the claims that follow below.
DETAILED DESCRIPTION
[0038] Throughout this application, the text refers to various
embodiments of the present nutrients, compositions, and methods.
The various embodiments described are meant to provide a variety of
illustrative examples and should not be construed as descriptions
of alternative species. Rather, it should be noted that the
descriptions of various embodiments provided herein may be of
overlapping scope. The embodiments discussed herein are merely
illustrative and are not meant to limit the scope of the present
invention.
[0039] Also throughout this disclosure, various publications,
patents and published patent specifications are referenced by an
identifying citation. The disclosures of these publications,
patents and published patent specifications are hereby incorporated
by reference into the present disclosure to more fully describe the
state of the art to which this invention pertains.
[0040] As used in the specification and claims, the singular form
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "an electrode"
includes a plurality of electrodes, including mixtures thereof.
[0041] As used herein, the term "comprising" is intended to mean
that the devices and methods include the recited components or
steps, but not excluding others. "Consisting essentially of when
used to define devices and methods, shall mean excluding other
components or steps of any essential significance to the
combination. "Consisting of" shall mean excluding other components
or steps. Embodiments defined by each of these transition terms are
within the scope of this invention.
[0042] All numerical designations, e.g., distance, size,
temperature, time, voltage and concentration, including ranges, are
approximations which are varied (+) or (-) by increments of 0.1. It
is to be understood, although not always explicitly stated that all
numerical designations are preceded by the term "about". It also is
to be understood, although not always explicitly stated, that the
components described herein are merely exemplary, and that
equivalents of such are known in the art.
Molecular Detection
[0043] The present disclosure provides methods and systems for
molecular detection and quantitation. In addition, the methods and
systems can also be configured to measure the affinity of a
molecule binding with another molecule. Further, such detection,
quantitation, and measurement can be carried out in a multiplexed
manner, greatly increasing its efficiency.
[0044] FIG. 1 provides an illustration of one embodiment of the
disclosed methods and systems. More specifically, the system
includes a ligand 104 that is capable of binding to a target
molecule 105 to be detected or quantitated. The ligand 104 can be
part of, or be linked to, a binding moiety (referred to as "binding
domain") 103 that is capable of binding to a specific binding motif
101 on a polymer scaffold 109. Together, the ligand 104 and the
binding domain 103 form a fusion molecule 102.
[0045] Therefore, if all present in a solution, the fusion molecule
102 binds, on one end, to a polymer scaffold 109 (or simply
"polymer") through the specific recognition and binding between the
binding motif 101 and the binding domain 103, and on the other end,
to the target molecule 105 by virtue of the interaction between the
ligand 104 and the target molecule 105. Such bindings cause the
formation of a complex that includes the polymer 109, the fusion
molecule 102 and the target molecule 105.
[0046] The formed complex can be detected by a device 108 that
includes a pore 107 that separates an interior space of the device
into two volumes, and a sensor adjacent to the pore 107 configured
to identify objects passing through the pore 107. This device is
referred throughout as a "nanopore". In some embodiments, the
nanopore 108 also includes means, such as electrodes connected to
power sources, for moving the polymer 109 from one volume to
another, across the pore 107. As the polymer 109 can be charged or
be modified to contain charges, one example of such means generates
a potential or voltage differential across the pore 107 to
facilitate and control the movement of the polymer 109.
[0047] When a sample that includes the formed complex is loaded to
the nanopore 108, the nanopore 108 can be configured to pass the
polymer 109 through the pore 107. When the binding motif 101 is
within the pore or adjacent to the pore 107, the binding status of
the motif 101 can be detected by the sensor.
[0048] The "binding status" of a binding motif, as used herein,
refers to whether the binding motif is bound to a fusion molecule
with a corresponding binding domain, and whether the fusion
molecule is also bound to a target molecule. Essentially, the
binding status can be one of three potential statuses: (i) the
binding motif is free and not bound to a fusion molecule (see 305
in FIG. 3); (ii) the binding motif is bound to a fusion molecule
that does not bind to a target molecule (see 306 in FIG. 3); or
(iii) the binding motif is bound to a fusion molecule that is bound
to a target molecule (see 307 in FIG. 3).
[0049] Detection of the binding status of a binding motif can be
carried out by various methods. In one aspect, by virtue of the
different sizes of the binding motif at each status, when the
binding motif passes through the pore, the different sizes result
in different electrical currents across the pore. In one aspect as
shown in FIG. 3, the measured current signals 301, when 305, 306,
and 307 pass through the pore, are signals 302, 303, and 304,
respectively. In this respect, no separate sensor is required for
the detection, as the electrodes, which are connected to power
sources and can detect the current, can serve the sensing function.
Either one or both of the electrodes, therefore, serve as a
"sensor."
[0050] In some aspects, an agent 106 as shown in FIG. 1 is added to
the complex to aid detection. This agent is capable to bind to the
target molecule or the ligand/target molecule complex. In one
aspect, the agent includes a charge, either negative or positive,
to facilitate detection. In another aspect, the agent adds size to
facilitate detection. In another aspect, the agent includes a
detectable label, such as a fluorophore.
[0051] In this context, an identification of status (iii) indicates
that a polymer-fusion molecule-target molecule complex has formed.
In other words, the target molecule is detected.
Particle Detection
[0052] The present disclosure also provides, in some aspects,
methods and systems for detecting, quantitating, and measuring
particles such as cells and microorganisms, including viruses,
bacteria, and cellular aggregates.
[0053] In some aspects, the pore that separates the device into two
volumes has a size that allows particles, such as viruses,
bacteria, cells, or cellular aggregates, to pass through. A ligand
that is capable of binding to a target particle to be detected or
quantitated can be included in the solution in the device such that
the ligand can bind to the unique target particle and the polymer
scaffold through a binding domain and a binding motif to form a
complex. Many such particles have unique markers on their surfaces
which can be specifically recognized by a ligand. For instance,
tumor cells can have tumor antigens expressed on the cell surface,
and bacterial cells can have endotoxins attached on the cell
membrane.
[0054] When the formed complex in a solution loaded into the
nanopore device is moved along with the polymer scaffold to pass
through the pore, the binding status of the complex within or
adjacent to the pore can be detected such that the target
microorganisms bound to the ligands can be identified using methods
similar to the molecular detection methods described elsewhere in
the disclosure.
Polymer Scaffold
[0055] A polymer scaffold suitable for use in the present
technology is a linear or linearized molecule, which has a length
that is several magnitudes greater than its width. Such a scaffold
can be loaded into a nanopore device and pass through the pore from
one end to the other.
[0056] Non-limiting examples of polymers include nuclei acids, such
as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or peptide
nucleic acid (PNA), and linearized proteins or peptides. In some
aspects, the DNA or RNA can be single-stranded or double-stranded,
or can be a DNA/RNA hybrid molecule.
[0057] In one aspect, the polymer is synthetic or chemically
modified. Chemical modification can help to stabilize the polymer,
add charges to the polymer to increase mobility, maintain
linearity, or add or modify the binding specificity. In some
aspects, the chemical modification is acetylation, methylation,
summolation, oxidation, phosphorylation, or glycosylation.
[0058] In some aspects, the polymer is electrically charged. DNA,
RNA, PNA and proteins are typically charged under physiological
conditions. Such polymers can be further modified to increase or
decrease the carried charge. Other polymers can be modified to
introduce charges. Charges on the polymer can be useful for driving
the polymer to pass through the pore of a nanopore device. For
instance, a charged polymer can move across the pore by virtue of
an application of voltage across the pore.
[0059] In some aspects, when charges are introduced to the polymer,
the charges can be added at the ends of the polymer. In some
aspects, the charges are spread over the polymer.
[0060] In one embodiment, each unit of the charged polymer is
charged at the pH selected. In another embodiment, the charged
polymer includes sufficient charged units to be pulled into and
through the pore by electrostatic forces. For example, a peptide
containing sufficient entities which can be charged at a selected
pH (lysine, aspartic acid, glutamic acid, etc.) so as to be used in
the devices and methods described herein is a charged polymer for
purposes of this invention. Likewise, a co-polymer comprising
methacrylic acid and ethylene is a charged polymer for the purposes
of this invention if there is sufficient charged carboxylate groups
of the methacrylic acid residue to be used in the devices and
methods described herein. In one embodiment, the charged polymer
includes one or more charged units at or close to one terminus of
the polymer. In another embodiment, the charged polymer includes
one or more charged units at or close to both termini of the
polymer. One co-polymer example is a DNA wrapped around protein
(e.g. DNA/nucleosome). Another example of a co-polymer is a
linearized protein conjugated to DNA at the N- and C-terminus.
Binding Motifs and Binding Domains
[0061] For nucleic acids and polypeptides such as the polymer
scaffold, a binding motif can be a nucleotide or peptide sequence
that is recognizable by a binding domain, which is typically a
functional portion of a protein, although a binding domain does not
have to be a peptide. For nucleic acids, for instance, there are
proteins that specifically recognize and bind to sequences (motifs)
such as promoters, enhancers, thymine-thymine dimers, and certain
secondary structures such as bent nucleotide and sequences with
single-strand breakage.
[0062] In some aspects, the binding motif includes a chemical
modification that causes or facilitates recognition and binding by
a binding domain. For example, methylated DNA sequences can be
recognized by transcription factors, DNA methyltransferases or
methylation repair enzymes.
[0063] Molecules, in particular proteins, that are capable of
specifically recognizing nucleotide binding motifs are known in the
art. For instance, protein domains such as helix-turn-helix, a zinc
finger, a leucine zipper, a winged helix, a winged helix turn
helix, a helix-loop-helix and an HMG-box, are known to be able to
bind to nucleotide sequences.
[0064] In some aspects, the binding domains can be locked nucleic
acids (LNAs), PNAs, transcription activator-like effector nucleases
(TALENs), clustered regularly interspaced short palindromic repeats
(CRISPRs), or aptamers.
Target Molecule/Particles and Ligands
[0065] In the present technology, a target molecule or particle is
detected or quantitated by virtue of its binding to a ligand in a
fusion molecule that binds to a polymer scaffold. A target molecule
or particle and a corresponding binding ligand can recognize and
bind each other. For a particle, there can be surface molecules or
markers suitable for a ligand to bind (therefore the marker and the
ligand form a binding pair).
[0066] Examples of binding pairs that enable binding between a
target molecule or a molecule on a particle include, but are not
limited to, antigen/antibody (or antibody fragment); hormone,
neurotransmitter, cytokine, growth factor or cell recognition
molecule/receptor; and ion or element/chelate agent or ion binding
protein, such as a calmodulin. The binding pairs can also be
single-stranded nucleic acids having complementary sequences,
enzymes and substrates, members of protein complex that bind each
other, enzymes and cofactors, nucleic acid/protein.
[0067] Therefore, any target molecule in need of detection or
quantitation, such as proteins, peptides, nucleic acids, chemical
compounds, ions, and elements, can find a corresponding binding
ligand. For the majority of proteins and nucleic acids, an antibody
or a complementary sequence can be readily prepared.
[0068] Likewise, binding ligands (such as antibodies) can be
readily found or prepared for particles, such as protein complexes
and protein aggregates, protein/nucleic acid complexes, fragmented
or fully assembled viruses, bacteria, cells, and cellular
aggregates.
Fusion Molecule
[0069] A "fusion molecule" is intended to mean a molecule or
complex that contains two functional regions, a binding domain and
a ligand. The binding domain is capable of binding to a binding
motif on a polymer scaffold, and the ligand is capable of binding
to a target molecule.
[0070] In some aspects, the fusion molecule is prepared by linking
the two regions with a bond or force. Such a bond and force can be,
for instance, a covalent bond, a hydrogen bond, an ionic bond, a
metallic bond, van der Walls force, hydrophobic interaction, or
planar stacking interaction.
[0071] In some aspects, the fusion molecule, such as a fusion
protein, can be expressed as a single molecule from a recombinant
coding nucleotide. In some aspects, the fusion molecule is a
natural molecule having a binding domain and a ligand suitable for
use in the present technology.
[0072] FIG. 2 illustrates a more specific embodiment of the system
shown in FIG. 1. In FIG. 2, the fusion molecule is a chimeric
protein that includes a zinc finger protein or domain 202 and a
human immunodeficiency virus (HIV) envelop protein 203. The zinc
finger protein 202 can bind to a suitable nucleotide sequence on
the polymer scaffold, a double-stranded DNA 201; the HIV envelop
protein 203 can bind to an anti-HIV antibody 204 which can be
present in a biological sample (e.g., a blood sample from a
patient) for detection.
[0073] When the double-stranded DNA 201 passes through a pore 205
of a nanopore device 206, the nanopore device 206 can detect
whether a fusion molecule is bound to the DNA 201 and whether the
bound fusion molecule binds to an anti-HIV antibody 204.
Measurement of Affinity of Binding
[0074] The present technology can be used also for measuring the
binding affinity between two molecules and to determine other
binding dynamics. For instance, after the binding motif passes
through the pore of a nanopore device, the device can be
reconfigured to reverse the moving direction of the polymer
scaffold (as described below) such that the binding motif can pass
through the pore again.
[0075] Before the binding motif enters the pore again, one can
change the conditions in the sample that is loaded into the
nanopore device. For instance, the condition can be one or more of
removing the target molecule from the sample, adding an agent that
competes with the target molecule or the ligand for binding, and
changing the pH, salt, or temperature.
[0076] Under the changed conditions, the binding motif passes
through the pore again. Therefore, whether the target molecule is
still bound to the fusion molecule can be detected to determine how
the changed conditions impact the binding.
[0077] In some aspects, once the binding motif is in the pore, it
is retained there while the conditions are changed, and thus the
impact of the changed conditions can be measured in situ.
[0078] Alternatively or in addition, the polymer scaffold can
include multiple binding motifs and each of the binding motifs can
bind to a fusion molecule which can bind to a target molecule or
particle. While each binding motif passes through the pore, the
conditions of the sample can be changed, allowing detection of
changed binding between the ligand and the target molecule or
particle on a continued basis.
Multiplexing
[0079] In some aspects, rather than including multiple binding
motif of the same kind as described above, a polymer scaffold can
include multiple types of binding motif, each can have different
corresponding binding domains. Meanwhile, a sample can include
multiple types of fusion molecules, each including one of such
binding domains and a ligand for a different target molecule or a
target microorganism.
[0080] With such a setting, a single polymer scaffold can be used
to detect multiple types of target molecules or target
microorganisms. FIG. 4 illustrates such a method. Here, a
double-stranded DNA 403 is used as the polymer scaffold, the
double-stranded DNA 403 including multiple binding motifs: two
copies of 404, two copies of 405, and one copy of 406.
[0081] When the DNA passes through a nanopore device 407 that has
two coaxial pores, 401 and 402, the binding status of each of the
binding motifs is detected, in which both copies of binding motif
404 bind to a corresponding target molecule, both copies of binding
motif 405 bind to a corresponding target molecule; and the fusion
molecule bound to binding motif 406 does not bind to a
corresponding target molecule.
[0082] This way, with a single polymer and a single nanopore
device, the present technology can detect different target
molecules or target microorganisms. Further, by determining how
many copies of binding motifs are bound to the target molecules or
target microorganisms, and by tuning conditions that impact the
bindings, the system can obtain more detailed binding dynamic
information.
Nanopore Devices
[0083] A nanopore device, as provided, includes at least a pore
that separates an interior space of the device into two volumes,
and at least a sensor adjacent to the pore configured to identify
objects passing through the pore.
[0084] The pore(s) in the nanopore device are of a nano scale. In
one aspect, each pore has a size that allows a small or large
molecule or microorganism to pass. In one aspect, each pore is at
least about 1 nm in diameter. Alternatively, each pore is at least
about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm,
12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25
nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm,
or 100 nm in diameter.
[0085] In one aspect, the pore is no more than about 100 nm in
diameter. Alternatively, the pore is no more than about 95 nm, 90
nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm,
40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, or 10 nm in diameter.
[0086] In some aspects, each pore is at least about 100 nm, 200 nm,
500 nm, 1000 nm, 2000 nm, 3000 nm, 5000 nm, 10000 nm, 20000 nm, or
30000 nm in diameter. In one aspect, the pore is no more than about
100000 nm in diameter. Alternatively, the pore is no more than
about 50000 nm, 40000 nm, 30000 nm, 20000 nm, 10000 nm, 9000 nm,
8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, or
1000 nm in diameter.
[0087] In one aspect, the pore has a diameter that is between about
1 nm and about 100 nm, or alternatively between about 2 nm and
about 80 nm, or between about 3 nm and about 70 nm, or between
about 4 nm and about 60 nm, or between about 5 nm and about 50 nm,
or between about 10 nm and about 40 nm, or between about 15 nm and
about 30 nm.
[0088] In some aspects, the pore(s) in the nanopore device are of a
larger scale for detecting large microorganisms or cells. In one
aspect, each pore has a size that allows a large cell or
microorganism to pass. In one aspect, each pore is at least about
100 nm in diameter. Alternatively, each pore is at least about 200
nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000
nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm,
1800 nm, 1900 nm, 2000 nm, 2500 nm, 3000 nm, 3500 nm, 4000 nm, 4500
nm, or 5000 nm in diameter.
[0089] In one aspect, the pore is no more than about 10000 nm in
diameter. Alternatively, the pore is no more than about 9500 nm,
9000 nm, 8500 nm, 8000 nm, 7500 nm, 7000 nm, 6500 nm, 6000 nm, 5500
nm, 5000 nm, 4500 nm, 4000 nm, 3500 nm, 3000 nm, 2500 nm, 2000 nm,
1500 nm, or 1000 nm in diameter.
[0090] In one aspect, the pore has a diameter that is between about
100 nm and about 10000 nm, or alternatively between about 200 nm
and about 9000 nm, or between about 300 nm and about 8000 nm, or
between about 400 nm and about 7000 nm, or between about 500 nm and
about 6000 nm, or between about 1000 nm and about 5000 nm, or
between about 1500 nm and about 3000 nm.
[0091] In some aspects, the nanopore device further includes means
to move a polymer scaffold to across the pore and/or means to
identify objects that pass through the pore. Further details are
provided below, when described in the context of a two-pore
device.
[0092] Compared to a single-pore nanopore device, a two-pore device
can be more easily configured to provide good control of speed and
direction of the movement of the polymer across the pores.
[0093] In one embodiment, the nanopore device includes a plurality
of chambers, each chamber in communication with an adjacent chamber
through at least one pore. Among these pores, two pores, namely a
first and a second pores, are placed so as to allow at least a
portion of a polymer to move out of the first pore and into the
second pore. Further, the device includes a sensor capable of
identifying the polymer during the movement. In one aspect, the
identification entails identifying individual components of the
polymer. Preferably, when a single sensor is employed, the single
sensor does not include two electrodes placed at both ends of a
pore to measure an ionic current across the pore.
[0094] In one aspect, the device includes three chambers connected
through two pores. Devices with more than three chambers can be
readily designed to include one or more additional chambers on
either side of a three-chamber device, or between any two of the
three chambers. Likewise, more than two pores can be included in
the device to connect the chambers.
[0095] In one aspect, there can be two or more pores between two
adjacent chambers, to allow multiple polymers to move from one
chamber to the next simultaneously. Such a multi-pore design can
enhance throughput of polymer analysis in the device.
[0096] In some aspects, the device further includes means to move a
polymer from one chamber to another. In one aspect, the movement
results in loading the polymer across both the first pore and the
second pore at the same time. In another aspect, the means further
enables the movement of the polymer, through both pores, in the
same direction.
[0097] For instance, in a three-chamber two-pore device (a
"two-pore" device), each of the chambers can contain an electrode
for connecting to a power supply so that a separate voltage can be
applied across each of the pores between the chambers.
[0098] In accordance with one embodiment of the present disclosure,
provided is a device comprising an upper chamber, a middle chamber
and a lower chamber, wherein the upper chamber is in communication
with the middle chamber through a first pore, and the middle
chamber is in communication with the lower chamber through a second
pore.
[0099] In some embodiments as shown in FIG. 5(I), the device
includes an upper chamber 505 (Chamber A), a middle chamber 504
(Chamber B), and a lower chamber 503 (Chamber C). The chambers are
separated by two separating layers or membranes (501 and 502) each
having a separate pore (511 or 512). Further, each chamber contains
an electrode (521, 522 or 523) for connecting to a power supply.
The annotation of upper, middle and lower chamber is in relative
terms and does not indicate that, for instance, the upper chamber
is placed above the middle or lower chamber relative to the ground,
or vice versa.
[0100] Each of the pores 511 and 512 independently has a size that
allows a small or large molecule or microorganism to pass. In one
aspect, each pore is at least about 1 nm in diameter.
Alternatively, each pore is at least about 2 nm, 3 nm, 4 nm, 5 nm,
6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm,
16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45
nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm in diameter.
[0101] In one aspect, the pore is no more than about 100 nm in
diameter. Alternatively, the pore is no more than about 95 nm, 90
nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm,
40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, or 10 nm in diameter.
[0102] In one aspect, the pore has a diameter that is between about
1 nm and about 100 nm, or alternatively between about 2 nm and
about 80 nm, or between about 3 nm and about 70 nm, or between
about 4 nm and about 60 nm, or between about 5 nm and about 50 nm,
or between about 10 nm and about 40 nm, or between about 15 nm and
about 30 nm.
[0103] In some aspects, the pore has a substantially round shape.
"Substantially round", as used here, refers to a shape that is at
least about 80 or 90% in the form of a cylinder. In some
embodiments, the pore is square, rectangular, triangular, oval, or
hexangular in shape.
[0104] Each of the pores 511 and 512 independently has a depth. In
one aspect, each pore has a depth that is least about 0.3 nm.
Alternatively, each pore has a depth that is at least about 0.6 nm,
1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm,
12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25
nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, or 90
nm.
[0105] In one aspect, each pore has a depth that is no more than
about 100 nm. Alternatively, the depth is no more than about 95 nm,
90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45
nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, or 10 nm.
[0106] In one aspect, the pore has a depth that is between about 1
nm and about 100 nm, or alternatively, between about 2 nm and about
80 nm, or between about 3 nm and about 70 nm, or between about 4 nm
and about 60 nm, or between about 5 nm and about 50 nm, or between
about 10 nm and about 40 nm, or between about 15 nm and about 30
nm.
[0107] In one aspect, the pores are spaced apart at a distance that
is between about 10 nm and about 1000 nm. In one aspect, the
distance is at least about 10 nm, or alternatively, at least about
20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150
nm, 200 nm, 250 nm, or 300 nm. In another aspect, the distance is
no more than about 1000 nm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm,
400 nm, 300 nm, 250 nm, 200 nm, 150 nm, or 100 nm.
[0108] In yet another aspect, the distance between the pores is
between about 20 nm and about 800 nm, between about 30 nm and about
700 nm, between about 40 nm and about 500 nm, or between about 50
nm and about 300 nm.
[0109] The two pores can be arranged in any position so long as
they allow fluid communication between the chambers and have the
prescribed size and distance between them. In one aspect, the pores
are placed so that there is no direct blockage between them. Still,
in one aspect, the pores are substantially coaxial, as illustrated
in FIG. 5(I).
[0110] In one aspect, as shown in FIG. 5(I), the device, through
the electrodes 521, 522, and 523 in the chambers 503, 504, and 505,
respectively, is connected to one or more power supplies. In some
aspects, the power supply includes a voltage-clamp or a patch-clamp
for supplying a voltage across each pore, which can also measure
the current through each pore independently. In this respect, the
power supply and the electrode configuration can set the middle
chamber to a common ground for both power supplies. In one aspect,
the power supplies are configured to apply a first voltage V.sub.1
between the upper chamber 505 (Chamber A) and the middle chamber
504 (Chamber B), and a second voltage V.sub.2 between the middle
chamber 504 and the lower chamber 503 (Chamber C).
[0111] In some aspects, the first voltage V.sub.1 and the second
voltage V.sub.2 are independently adjustable. In one aspect, the
middle chamber is adjusted to be a ground relative to the two
voltages. In one aspect, the middle chamber comprises a medium for
providing conductance between each of the pores and the electrode
in the middle chamber. In one aspect, the middle chamber comprises
a medium for providing a resistance between each of the pores and
the electrode in the middle chamber. Keeping such a resistance
sufficiently small relative to the nanopore resistances is useful
for decoupling the two voltages and currents across the pores,
which is helpful for the independent adjustment of the
voltages.
[0112] Adjustment of the voltages can be used to control the
movement of charged particles in the chambers. For instance, when
both voltages are set in the same polarity, a properly charged
particle can be moved from the upper chamber to the middle chamber
and to the lower chamber, or the other way around, sequentially. In
some aspects, when the two voltages are set to opposite polarity, a
charged particle can be moved from either the upper or the lower
chamber to the middle chamber and kept there.
[0113] The adjustment of the voltages in the device can be
particularly useful for controlling the movement of a large
molecule, such as a charged polymer, that is long enough to cross
both pores at the same time. In such an aspect, the direction and
the speed of the movement of the molecule can be controlled by the
relative magnitude and polarity of the voltages as described
below.
[0114] The device can contain materials suitable for holding liquid
samples, in particular, biological samples, and/or materials
suitable for nanofabrication. In one aspect, such materials include
dielectric materials such as, but not limited to, silicon, silicon
nitride, silicon dioxide, graphene, carbon nanotubes, TiO.sub.2,
HfO.sub.2, Al.sub.2O.sub.3, or other metallic layers, or any
combination of these materials. In some aspects, for example, a
single sheet of graphene membrane of about 0.3 nm thick can be used
as the pore-bearing membrane.
[0115] Devices that are microfluidic and that house two-pore
microfluidic chip implementations can be made by a variety of means
and methods. For a microfluidic chip comprised of two parallel
membranes, both membranes can be simultaneously drilled by a single
beam to form two concentric pores, though using different beams on
each side of the membranes is also possible in concert with any
suitable alignment technique. In general terms, the housing ensures
sealed separation of Chambers A-C. In one aspect as shown in FIG.
5(II), the housing would provide minimal access resistance between
the voltage electrodes 521, 522, and 523 and the nanopores 511 and
512, to ensure that each voltage is applied principally across each
pore.
[0116] In one aspect, the device includes a microfluidic chip
(labeled as "Dual-core chip") comprising two parallel membranes
connected by spacers. Each membrane contains a pore drilled by a
single beam through the center of the membrane. Further, the device
preferably has a Teflon.RTM. housing for the chip. The housing
ensures sealed separation of Chambers A-C and provides minimal
access resistance for the electrode to ensure that each voltage is
applied principally across each pore.
[0117] More specifically, the pore-bearing membranes can be made
with transmission electron microscopy (TEM) grids with a 5-100 nm
thick silicon, silicon nitride, or silicon dioxide windows. Spacers
can be used to separate the membranes, using an insulator, such as
SU-8, photoresist, PECVD oxide, ALD oxide, ALD alumina, or an
evaporated metal material, such as Ag, Au, or Pt, and occupying a
small volume within the otherwise aqueous portion of Chamber B
between the membranes. A holder is seated in an aqueous bath that
comprises the largest volumetric fraction of Chamber B. Chambers A
and C are accessible by larger diameter channels (for low access
resistance) that lead to the membrane seals.
[0118] A focused electron or ion beam can be used to drill pores
through the membranes, naturally aligning them. The pores can also
be sculpted (shrunk) to smaller sizes by applying a correct beam
focusing to each layer. Any single nanopore drilling method can
also be used to drill the pair of pores in the two membranes, with
consideration to the drill depth possible for a given method and
the thickness of the membranes. Predrilling a micro-pore to a
prescribed depth and then a nanopore through the remainder of the
membranes is also possible to further refine the membrane
thickness.
[0119] In another aspect, the insertion of biological nanopores
into solid-state nanopores to form a hybrid pore can be used in
either or both pores in the two-pore method. The biological pore
can increase the sensitivity of the ionic current measurements, and
is useful when only single-stranded polynucleotides are to be
captured and controlled in the two-pore device, e.g., for
sequencing.
[0120] By virtue of the voltages present at the pores of the
device, charged molecules can be moved through the pores between
chambers. Speed and direction of the movement can be controlled by
the magnitude and polarity of the voltages. Further, because each
of the two voltages can be independently adjusted, the direction
and speed of the movement of a charged molecule can be finely
controlled in each chamber.
[0121] One example concerns a charged polymer scaffold, such as a
DNA, having a length that is longer than the combined distance that
includes the depth of both pores plus the distance between the two
pores. For example, a 1000 bp dsDNA is about 340 nm in length, and
would be substantially longer than the 40 nm spanned by two 10
nm-deep pores separated by 20 nm. In a first step, the
polynucleotide is loaded into either the upper or the lower
chamber. By virtue of its negative charge under a physiological
condition at a pH of about 7.4, the polynucleotide can be moved
across a pore on which a voltage is applied. Therefore, in a second
step, two voltages, in the same polarity and at the same or similar
magnitudes, are applied to the pores to move the polynucleotide
across both pores sequentially.
[0122] At about time when the polynucleotide reaches the second
pore, one or both of the voltages can be changed. Since the
distance between the two pores is selected to be shorter than the
length of the polynucleotide, when the polynucleotide reaches the
second pore, it is also in the first pore. A prompt change of
polarity of the voltage at the first pore, therefore, will generate
a force that pulls the polynucleotide away from the second pore as
illustrated in FIG. 5(III).
[0123] Assuming that the two pores have identical voltage-force
influence and |V.sub.1|=|V.sub.2|+.delta.V, the value .delta.V>0
(or <0) can be adjusted for tunable motion in the V.sub.1 (or
V.sub.2) direction. In practice, although the voltage-induced force
at each pore will not be identical with V.sub.1=V.sub.2,
calibration experiments can identify the appropriate bias voltage
that will result in equal pulling forces for a given two-pore chip;
and variations around that bias voltage can then be used for
directional control.
[0124] If, at this point, the magnitude of the voltage-induced
force at the first pore is less than that of the voltage-induced
force at the second pore, then the polynucleotide will continue
crossing both pores towards the second pore, but at a lower speed.
In this respect, it is readily appreciated that the speed and
direction of the movement of the polynucleotide can be controlled
by the polarities and magnitudes of both voltages. As will be
further described below, such a fine control of movement has broad
applications.
[0125] Accordingly, in one aspect, provided is a method for
controlling the movement of a charged polymer through a nanopore
device. The method entails (a) loading a sample comprising a
charged polymer in one of the upper chamber, middle chamber or
lower chamber of the device of any of the above embodiments,
wherein the device is connected to power supplies for providing a
first voltage between the upper chamber and the middle chamber, and
a second voltage between the middle chamber and the lower chamber;
(b) setting an initial first voltage and an initial second voltage
so that the polymer moves between the chambers, thereby locating
the polymer across both the first and second pores; and (c)
adjusting the first voltage and the second voltage so that both
voltages generate force to pull the charged polymer away from the
middle chamber (voltage-competition mode), wherein the two voltages
are different in magnitude, under controlled conditions, so that
the charged polymer moves across both pores in either direction and
in a controlled manner.
[0126] To establish the voltage-competition mode in step (c), the
relative force exerted by each voltage at each pore is to be
determined for each two-pore device used, and this can be done with
calibration experiments by observing the influence of different
voltage values on the motion of the polynucleotide, which can be
measured by sensing known-location and detectable features in the
polynucleotide, with examples of such features detailed later in
this disclosure. If the forces are equivalent at each common
voltage, for example, then using the same voltage value at each
pore (with common polarity in upper and lower chambers relative to
grounded middle chamber) creates a zero net motion in the absence
of thermal agitation (the presence and influence of Brownian motion
is discussed below). If the forces are not equivalent at each
common voltage, achieving equal forces involves the identification
and use of a larger voltage at the pore that experiences a weaker
force at the common voltage. Calibration for voltage-competition
mode can be done for each two-pore device, and for specific charged
polymers or molecules whose features influence the force when
passing through each pore.
[0127] In one aspect, the sample containing the charged polymer is
loaded into the upper chamber and the initial first voltage is set
to pull the charged polymer from the upper chamber to the middle
chamber and the initial second voltage is set to pull the polymer
from the middle chamber to the lower chamber. Likewise, the sample
can be initially loaded into the lower chamber, and the charged
polymer can be pulled to the middle and the upper chambers.
[0128] In another aspect, the sample containing the charged polymer
is loaded into the middle chamber; the initial first voltage is set
to pull the charged polymer from the middle chamber to the upper
chamber; and the initial second voltage is set to pull the charged
polymer from the middle chamber to the lower chamber.
[0129] In one aspect, the adjusted first voltage and second voltage
at step (c) are about 10 times to about 10,000 times as high, in
magnitude, as the difference between the two voltages. For
instance, the two voltages can be 90 mV and 100 mV, respectively.
The magnitude of the two voltages, about 100 mV, is about 10 times
of the difference between them, 10 mV. In some aspects, the
magnitude of the voltages is at least about 15 times, 20 times, 25
times, 30 times, 35 times, 40 times, 50 times, 100 times, 150
times, 200 times, 250 times, 300 times, 400 times, 500 times, 1000
times, 2000 times, 3000 times, 4000 times, 5000 times, 6000 times,
7000 times, 8000 times or 9000 times as high as the difference
between them. In some aspects, the magnitude of the voltages is no
more than about 10000 times, 9000 times, 8000 times, 7000 times,
6000 times, 5000 times, 4000 times, 3000 times, 2000 times, 1000
times, 500 times, 400 times, 300 times, 200 times, or 100 times as
high as the difference between them.
[0130] In one aspect, real-time or on-line adjustments to the first
voltage and the second voltage at step (c) are performed by active
control or feedback control using dedicated hardware and software,
at clock rates up to hundreds of megahertz. Automated control of
the first or second or both voltages is based on feedback of the
first or second or both ionic current measurements.
Sensors
[0131] In one aspect, the nanopore device further includes one or
more sensors to carry out the identification of the binding status
of the binding motifs.
[0132] The sensors used in the device can be any sensor suitable
for identifying a molecule or particle, such as a polymer. For
instance, a sensor can be configured to identify the polymer by
measuring a current, a voltage, a pH value, an optical feature, or
residence time associated with the polymer or one or more
individual components of the polymer. In one aspect, the sensor
includes a pair of electrodes placed at two sides of a pore to
measure an ionic current across the pore when a molecule or
particle, in particular a polymer, moves through the pore.
[0133] In one embodiment, the sensor measures an optical feature of
the polymer or a component (or unit) of the polymer. One example of
such measurement includes the identification of an absorption band
unique to a particular unit by infrared (or ultraviolet)
spectroscopy.
[0134] When residence time measurements are used, the size of the
unit can be correlated to the specific unit based on the length of
time it takes to pass through the sensing device.
[0135] Still further, the sensor can include an enzyme distal to
that sensor, which enzyme is capable of separating the terminal
unit of the polymer from the penultimate unit thereby providing for
a single molecular unit of the polymer. The single molecule, such
as a single nucleotide or an amino acid, can then be detected with
methods such as mass spectrometry. Methods for measuring such a
single unit are known in the art and include those developed by Cal
Tech (see, e.g.,
spectrum.ieee.org/tech-talk/at-work/test-and-measurement/a-scale-for-weig-
hing-single-molecules). The results of such analysis can be
compared to those of the sensing device to confirm the correctness
of the analysis.
[0136] In some embodiments, the sensor is functionalized with
reagents that form distinct non-covalent bonds with each DNA base.
In this respect, a gap formed by the sensor can be larger and still
allow effective measuring. For instance, when used with a sensor
functionalized with reagents, a 2.5 nm gap can be as effective as a
0.8 nm gap.
[0137] Tunnel sensing with a functionalized sensor is termed
"recognition tunneling." Using a Scanning Tunneling Microscope
(STM) with recognition tunneling, a DNA base flanked by other bases
in a short DNA oligomer can be identified. Recognition tunneling
can also provide a "universal reader" designed to hydrogen-bond to
each of the four DNA bases (A, C, G, T) in a unique orientation,
and also to the base 5-methyl-cytosine (mC) which is naturally
occurring due to epigenetic modifications.
[0138] A limitation with the conventional recognition tunneling is
that it can detect only freely diffusing DNA that randomly binds in
the gap, or that happens to be in the gap during microscope motion,
with no method of explicit capture to the gap. However, the
collective drawbacks of the STM setup can be eliminated when the
recognition reagent, once optimized for sensitivity, is
incorporated within an electrode tunneling gap in a nanopore
channel.
[0139] Accordingly, in one embodiment, the sensor comprises surface
modification by a reagent. In one aspect, the reagent is capable of
forming a non-covalent bond with a nucleotide. In a particular
aspect, the bond is a hydrogen bond. Non-limiting examples of the
reagent include 4-mercaptobenzamide and
1-H-Imidazole-2-carboxamide.
[0140] A significant advantage of the methods in the present
technology is that they can be engineered, in principle, to provide
direct tracking of progress through homopolymeric regions (base
repeats). Tracking repeats is useful, for example, since deletions
and insertions of specific mononucleotide repeats (7, 9 nt) within
human mitochondrial DNA have been implicated in several types of
cancer. Direct base repeat tracking is difficult, if not
impossible, with ionic current sensing.
[0141] In ionic current sensing, there is no distinct
signal-per-nucleotide of motion of homopolymeric ssDNA through the
pore. Therefore, an ideal nanopore sequencing platform may include
an auxiliary sensing method that can track per-nucleotide motion
progress while also achieving single-nucleotide sensitivity.
Transitions between neighboring nucleotides in oligomers can be
observable with recognition tunneling, making it a candidate for
sequencing that permits direct base-repeat tracking.
[0142] Therefore, the methods of the present disclosure can provide
DNA delivery rate control for one or more recognition tunneling
sites, each positioned in one or both of the nanopore channels, and
voltage control can ensure that each nucleotide resides in each
site for a sufficient duration for robust identification.
[0143] Sensors in the devices and methods of the present disclosure
can comprise gold, platinum, graphene, or carbon, or other suitable
materials. In a particular aspect, the sensor includes parts made
of graphene. Graphene can act as a conductor and an insulator, thus
tunneling currents through the graphene and across the nanopore can
sequence the translocating DNA.
[0144] In some embodiments, the tunnel gap has a width from about 1
nm to about 20 nm. In one aspect, the width of the gap is at least
about 1 nm, or alternatively, at least about 1.5, 2, 2.5, 3, 3.5,
4, 4.5, 5, 6, 7, 8, 9, 10, 12, or 15 nm. In another aspect, the
width of the gap is not greater than about 20 nm, or alternatively,
not greater than about 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,
8, 7, 6, 5, 4, 3, or 2 nm. In some aspects, the width is between
about 1 nm and about 15 nm, between about 1 nm and about 10 nm,
between about 2 nm and about 10 nm, between about 2.5 nm and about
10 nm, or between about 2.5 nm and about 5 nm.
[0145] In some embodiments, the sensor is an electric sensor. In
some embodiments, the sensor detects a fluorescent detection means
when the target molecule or the detectable label passing through
has a unique fluorescent signature. A radiation source at the
outlet of the pore can be used to detect that signature.
[0146] It is to be understood that while the invention has been
described in conjunction with the above embodiments, the foregoing
description and examples are intended to illustrate and not limit
the scope of the invention. Other aspects, advantages and
modifications within the scope of the invention will be apparent to
those skilled in the art to which the invention pertains.
* * * * *